EP0659276B1 - Immunoassay for detecting collagen or collagen fragments - Google Patents
Immunoassay for detecting collagen or collagen fragments Download PDFInfo
- Publication number
- EP0659276B1 EP0659276B1 EP93917678A EP93917678A EP0659276B1 EP 0659276 B1 EP0659276 B1 EP 0659276B1 EP 93917678 A EP93917678 A EP 93917678A EP 93917678 A EP93917678 A EP 93917678A EP 0659276 B1 EP0659276 B1 EP 0659276B1
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- EP
- European Patent Office
- Prior art keywords
- collagen
- sequence
- type
- antigen
- peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
Definitions
- the invention relates to antigens for the production of antibodies against type I collagen, a process for the production of such antigens, antibodies against type I collagen which Immunization available with an antigen according to the invention are, and the use of such antibodies for detection of collagen type I.
- Collagen is an important structural protein in the connective tissue of skin, cartilage and bone.
- a characteristic Property of mature collagen, such as particular tissues Bone, or cartilage is the cross-linking of fibers lying side by side by hydroxylysylpyridinoline or lysylpyridinoline (D. Fujimoto et al., J. Biochem 83 (1978), 863-867; D.
- a disadvantage of this isolated from natural sources Peptides is not a sure source of reproducible Production of the antigens or binding partners present in the test is.
- Another disadvantage of natural sources isolated peptides is at risk of contamination infectious material.
- antigens can e.g. through chemical synthesis of a peptide which is an epitope of the antigen corresponds.
- small peptides with a Molecular weight of about 700 - 1500 D used that is Binding to a carrier molecule is required to use an antigen to obtain immunogenic effect. By binding to that The carrier molecule must not change the structure of the epitope will. The coupling to the carrier molecule therefore takes place previously preferred at the ends of the peptide chain in sufficient distance from the suspected epitope area (Laboratory Technics in Biochemistry and Molecular Biology, Synthetic Polypeptides as Antigens, Editors R.H. Burdon and P.H. van Knippenberg, Elsevier, Amsterdam, New York, Oxford 1988, pages 95-100).
- a problem in the chemical synthesis of a defined Antigen which is a natural degradation product of cross-linked Corresponds to collagen, which represents the cross-linking resulting hydroxylysyl or lysylpyridinoline structure whose chemical synthesis has so far not been successful is.
- the object of the invention was therefore to create a defined antigen for the production of antibodies against type I collagen or a collagen fragment of which, for use as a specific binding partner of Antibody to collagen or collagen fragments in one competitive immunoassay and as standard material for preparation a standard or calibration curve in a competitive Immunoassay for the detection of collagen or type I collagen fragments to provide.
- the object of the invention is therefore a competitive immunoassay for the detection of collagen type I or collagen fragments in a sample, which is characterized in that a binding partner, which contains a synthetic linear peptide which a sequence of the non-helical linear C- or N-terminal Region of type I collagen, with an antibody, which is able to bind the synthetic linear peptide, and the Incubated sample and binding of the antibody to the binding partner is determined in a suitable manner.
- Another object of the invention is a standard material to create a standard or calibration curve in a competitive immunoassay for the detection of collagen or Type I collagen fragments, characterized in that an antigen is contained, which is a synthetic peptide, which one Sequence of the non-helical C- or N-terminal region of Corresponds to type I collagen.
- Another object of the invention is also an antigen for the production of antibodies against type I or collagen Collagen fragments thereof, which is a synthetic linear peptide, which a sequence of the non-helical C or N-terminal region of collagen type I contains, as well as contains with this Antigen produced antibodies.
- the concentration of collagen breakdown products is an important diagnostic marker for the extent of osteolysis.
- synthetic linear peptides it is possible to carry out a competitive immunoassay for the detection of collagen or collagen fragments. It has surprisingly been found that these peptides compete very well with the collagen fragments that occur in natural samples such as plasma, serum or urine for antibodies against these collagen fragments and thus enable a competitive test.
- Such antibodies against collagen or collagen breakdown products are commercially available, for example in the telopeptide ICTP [ 125 I) radioimmunoassay kit from Orion Diagnostica, Finland. However, they can also be produced according to the invention using the synthetic linear peptide.
- WO-A-90/08195 describes a test for the detection of type IX collagen, by a monoclonal antibody, which was produced by means of synthetic peptides which represent sequences of the globular domain NC 4.
- the situation in collagen IX cannot be compared with type I collagen; in particular, no characteristic cross-linking occurs in the globular domain in type IX collagen.
- EP-A-0298210 a peptide sequence is used to immunize animals in order to raise antibodies which recognize type III procollagen. Procollagen is not a diagnostic marker for osteolysis.
- the synthetic linear peptide directly as a binding partner that connects to a Solid phase is bound, used or coupled to a second component.
- the coupling to the second component is preferably carried out via the N- and C-terminal amino acids of the linear peptide.
- between the peptide and a spacer can be inserted into the second component.
- the second component can serve, for example, the peptide to couple indirectly to a solid phase. examples for this are known to the expert.
- the peptide is preferred on bovine serum albumin coupled and the coupling product adsorptively a solid phase, for example a plastic tube.
- the peptide can also be covalently bound to biotin.
- the Attachment to the solid phase then takes place via the bond to Avidin or streptavidin, which in turn adhere to the solid phase was bound.
- the second component can also be used as a carrier serve for multiple peptides, for example in a competitive turbidimetric inhibition immunoassay (TINIA), where several peptides on for example albumin, immunoglobulins, ⁇ -galactosidase, polymers such as polylysine or dextran molecules as described in EP-A-0 545 350 or particles how latex are coupled. Preferably 30 to 40 Peptide molecules coupled per carrier molecule.
- the peptide can also coupled to a component that represents a marker be. Examples of a11 these test variants are the Known specialist.
- the antibody can be used simultaneously or during the test sequentially with the sample and the binding partner who Contains synthetic linear peptide to be incubated. Subsequently is the amount of bound in the usual way or unbound antibody.
- a competitive Test variants for determining the amount of bound or unbound antibody can, for example, agglutination tests, such as TINIA, or FPIA (fluorescence polarization immunoassay) (W. Dandliker et al., J.Exp. Med.
- the peptides according to the invention have proven to be special suitable for use as a defined binding partner for Competition with the sample for binding to the antibodies proven.
- the synthetic linear ones are particularly preferred Peptide with the sequence shown in SEQ ID NO 1, 2 or 3.
- collagen breakdown products can be isolated from natural Material can be used. However, these stand out naturally by a certain fluctuation. More suitable An antigen that has proven to be the standard material contains synthetic linear peptide according to the invention.
- the Antigen of the standard can only be made from this peptide consist of or from this peptide attached to a suitable Carrier is coupled, for example, for better water solubility of the peptide.
- the peptide and carrier become the linear peptide, the a sequence of the non-helical C or N-terminal region of type I collagen, synthesized and via its N- or C-terminal amino acid using suitable coupling methods bound to the carrier molecule. It can be a or several peptides are bound per carrier molecule.
- the coupling can take place via a spacer.
- a spacer For certain Purposes, such as for agglutination tests, it may be advantageous to use several peptides according to the invention different sequence to bind to a carrier molecule especially if polyclonal antibodies are used in the test, which are not produced with the aid of the antigen according to the invention and usually recognize several epitopes.
- Antibodies with the help are also suitable of an antigen which is the linear synthetic according to the invention Contains peptide were obtained.
- the linear synthetic peptides that of one or more sequences of the non-helical C- or N-terminal region of collagen type I correspond to a suitable one Carrier protein such as keyhole limpet hemocyanins, Bovine serum albumin or edestin can be bound.
- Carrier protein such as keyhole limpet hemocyanins, Bovine serum albumin or edestin
- To produce these antigens or immunogens are first the linear peptides chemically in the usual way synthesized. The synthetic is then coupled Peptides via the N-terminal amino group Maleinimidohexanoic acid N-hydroxysucciminide ester to the above called carrier proteins. It has surprisingly turned out to be shown that for the production of antibodies for the competitive test management are suitable, synthetic linear Peptides with the sequence shown in SEQ ID NO 1, 2 or 3 are particularly suitable.
- antigens according to the invention which is a synthetic linear peptide containing a sequence of the non-helical Corresponds to the C- or N-terminal region of collagen type I, it is possible to get antibodies that do not only the peptide according to the invention but also those in body fluids Detect occurring breakdown products of collagen.
- Another object of the invention is therefore a method for the production of antibodies against collagen or collagen fragments by immunization with an inventive Antigen and isolation of the desired antibody from the Serum of the immunized animals according to known methods.
- the desired antibody is isolated via immunoadsorption on a carrier protein, preferably Sepharose, coupled peptide of the SEQ ID NO 1, 2 or 3 sequence shown.
- a preferred subject of the invention is a method for the production of monoclonal antibodies against collagen or Collagen fragments by immunization with an inventive Antigen, immortalization of the spleen cells of the immunized Animals, cloning of those immortalized spleen cells, which produce the desired antibody and Isolation of the antibody from the cloned cells or the Culture supernatant of these cells.
- the immunization takes place in the commonly used for this Animals, preferably mice or rabbits used.
- Immortalization of the spleen cells of the immunized animals takes place according to methods familiar to the person skilled in the art, e.g. of the Hybridoma technology (Köhler and Milstein, Nature 256 (1975), 495 - 497) or by transformation with the Epstein-Barr virus (EBV transformation).
- Hybridoma technology Kerterion and Milstein, Nature 256 (1975), 495 - 497
- EBV transformation Epstein-Barr virus
- Another object of the invention are those according to the invention Polyclonal and monoclonal methods available Antibody.
- the antibodies of the invention can therefore in the above described detection methods for the determination of collagen or collagen fragments can be used.
- Another object of the invention is therefore the use of a polyclonal or monoclonal according to the invention Antibody for the determination of osteolysis by incubation of the Antibody with a tissue sample and determination of the to the Antibody binding collagen breakdown product.
- the coupling times were 30 and 40 minutes.
- the split time was 20 minutes and was with a 50% solution Piperidine performed in the DMF.
- the washing steps were with DMF performed eight times after each reaction step.
- the peptide was released by treatment of the Filtered off solvent and with dichloromethane and methanol washed resin with 1 ml of a solution of 90% trifluoroacetic acid, 3% thioanisole, 3% ethanedithiol and 3% thiocresol within 20 minutes and 140 minutes.
- the product was combined by adding 15 ml of cold diisopropyl ether Filtrate precipitated and isolated by filtration. The residue was dissolved in 50% acetic acid and lyophilized. 8 mg of white lyophilizate were of a purity obtained by 79% according to HPLC. The identity was determined using FAB mass spectroscopy approved.
- the peptide produced according to example la) is by acylation with maleimidohexanoyl-N-hydroxysuccinimide (MHS) activated.
- MHS maleimidohexanoyl-N-hydroxysuccinimide
- 0.1 mmol of the peptide in 20 ml becomes 0.1 mol / l Potassium phosphate buffer pH 7.5 dissolved, with a solution of 0.1 mmol of MHS in 6 ml of dioxane and 20 min. stirred at 20 ° C. Then the pH is adjusted to pH 4 with glacial acetic acid adjusted and the reaction mixture immediately lyophilized.
- the lyophilisate is dissolved in 5 ml of water and preparative HPLC on a Waters Delta-Pak® C18 column (100 ⁇ , 15 ⁇ m 50 x 300 mm) over an elution gradient of 100% A. (Water 0.1% trifluoroacetic acid) to 100% B (99.9% acetonitrile 0.1% trifluoroacetic acid).
- a carrier protein is thus obtained, one versus the other native state has increased number of SH groups. Therefor becomes a solution of 1.39 g KLH in 500 ml 0.1 mol / l Potassium phosphate buffer pH 8.5 within 20 min. 113.51 mg SATP (dissolved in 10 ml of dioxane) was added dropwise. After 30 min. stir at 20 ° C the pH of the reaction solution becomes 0.1 mol / l Sodium hydroxide solution adjusted to pH 8.5 and another 24 hours touched.
- the solution is then made using an Amicon cell (Membrane YM10) concentrated to 100 ml, 3 x 24 Hours against 3 1 0.1 mol / l potassium phosphate buffer pH 8.5 / 0.05 mol / l sodium chloride dialyzed and then lyophilized.
- Amicon cell Membrane YM10
- the immunogens contained that Peptide with the sequence mentioned in SEQ ID NO 2, which the Amino acids Nos. 892 to 907 in the sequence of the ⁇ chain of Corresponds to type I collagen. KLH or ⁇ -galactosidase.
- the animals were monthly with the immunogens in complete Freund's adjuvant immunized. The dose was 500 ⁇ g per animal and immunization. Blood samples were taken four months after first immunization removed and the antibodies obtained in response to Collagen fragments checked.
- the wells of the titer plates were each 100 ⁇ l Solution filled, the 10 ⁇ g / ml collagen fragments in coating buffer contained.
- the collagen fragments were made accordingly the information in EP-A-0 505 210 by protease digestion made from human collagen from bones. After one hour incubation at room temperature with shaking was washed three times with washing solution.
- the antisera were diluted 1: 4000 with incubation buffer and 100 ⁇ l each in the wells of the microtiter plate Incubated for one hour with shaking at room temperature. Subsequently the wells were washed three times with wash solution washed.
- a conjugate of horseradish peroxidase with rabbit antibodies against the Fc part of sheep IgG is in the incubation buffer diluted to a concentration of 12.5 mU / ml and the Wells of the microtiter plate are loaded with 100 ⁇ l each. After an hour of incubation with shaking at room temperature the titer plates are washed three times with washing solution.
- the wells of a 96-well microtiter plate are made according to EP-A 0 344 578 with streptavidin (100 ul of a solution of 1 ⁇ g / ml in PBS) coated at 4 ° C overnight and still free nonspecific binding sites by incubation with 300 ⁇ l BSA (Bovine serum albumin, 10 mg / ml) for 2 hours at room temperature blocked.
- streptavidin 100 ul of a solution of 1 ⁇ g / ml in PBS coated at 4 ° C overnight and still free nonspecific binding sites by incubation with 300 ⁇ l BSA (Bovine serum albumin, 10 mg / ml) for 2 hours at room temperature blocked.
- BSA Bovine serum albumin
- the biotinylated peptide is in PBS, 0.05% Tween 20, 1% BSA dissolved in a concentration of 10ng / ml and by incubating 100 ⁇ l per well for 1 hour the microtiter plate coated with streptavidin bound. Then unbound peptide by three times Wash with PBS, 0.05% Tween 20 removed.
- each of the sample to be examined (serum, plasma or a standard) with 150 ul of the invention Antibody according to Example 4 for 2 hours at 37 ° C (or above Incubated at 4 ° C). 100 ⁇ l of this approach each are bound into the wells of the microtiter plate Decapeptide added and incubated at 37 ° C for 60 minutes. Only the one after incubation with the sample can unbound antibody excess of the antiserum bind the immobilized decapeptide.
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Abstract
Description
Die Erfindung betrifft Antigene zur Herstellung von Antikörpern gegen Kollagen Typ I, ein Verfahren zur Herstellung von solchen Antigenen, Antikörper gegen Kollagen Typ I welche durch Immunisierung mit einem erfindungsgemäßen Antigen erhältlich sind, sowie die Verwendung solcher Antikörper zum Nachweis von Kollagen Typ I.The invention relates to antigens for the production of antibodies against type I collagen, a process for the production of such antigens, antibodies against type I collagen which Immunization available with an antigen according to the invention are, and the use of such antibodies for detection of collagen type I.
Kollagen stellt ein wichtiges Strukturprotein im Bindegewebe von Haut, Knorpel und Knochen dar. Bekannt sind 11 Typen, die jeweils aus drei Ketten bestehen. Jeder Typ setzt sich aus 1 - 3 verschiedenen Ketten zusammen, die als α1, α2 und α3 bezeichnet werden (E. Miller et al. in Methods in Enzymology 144, Structural and Contractile Proteins, ed. L. Cunningham, Academic Press Inc. 1987, p. 3 - 41). Eine charakteristische Eigenschaft des reifen Kollagens bestimmter Gewebe, wie insbesondere Knochen oder Knorpel, ist die Quervernetzung von nebeneinander liegenden Fasern durch Hydroxylysylpyridinolin oder Lysylpyridinolin (D. Fujimoto et al., J. Biochem 83 (1978), 863 - 867; D. Eyre et al., Ann. Rev. Biochem 53 (1984), 717-748 und D. Eyre, Methods in Enzymology 144 (1987), 115-139). Diese Quervernetzungen können als biochemischer Marker für den spezifischen Nachweis von Kollagen genutzt werden (Z. Gunja-Smith et al., Biochem. J. 197 (1981), 759-762). Beim Abbau von extrazellulärem Kollaqen gelangen Hydroxylysylpyridinolin- oder Lysylpyridinolin-derivate, welche Peptidseitenketten enthalten oder freie Pyridinolin-Derivate mit Lysyl- oder Hydroxylysylresten, wie in der WO 91/10141 beschrieben, in Körperflüssigkeiten wie Blut oder Urin. Der Nachweis dieser Verbindungen in Körperflüssigkeiten ergibt daher Hinweise auf den Abbau von extrazellulärem Kollagen, wie er z.B. bei der Osteoporose sowie infolge von Tumoren des Knochengewebes auftritt. Zum Nachweis von solchen Hydroxylysyl- oder Lysylpyridinolinen mit Peptidseitenketten wurden in der WO 89/12824 monoklonale Antikörper beschrieben, welche durch Immunisierung mit entsprechenden quervernetzten Kollagenfragmenten erhalten wurden, die aus dem Urin isoliert werden können. Auch bei dem in WO 91/08478 beschriebenen Verfahren erfolgt der Nachweis von Kollagen über einen Antikörper gegen natürliche, d.h. in vivo erzeugte quervernetzte Abbauprodukte von Kollagen.Collagen is an important structural protein in the connective tissue of skin, cartilage and bone. There are 11 types known each consist of three chains. Every type is exposed 1 - 3 different chains together, called α1, α2 and α3 (E. Miller et al. in Methods in Enzymology 144, Structural and Contractile Proteins, ed. L. Cunningham, Academic Press Inc. 1987, p. 3 - 41). A characteristic Property of mature collagen, such as particular tissues Bone, or cartilage, is the cross-linking of fibers lying side by side by hydroxylysylpyridinoline or lysylpyridinoline (D. Fujimoto et al., J. Biochem 83 (1978), 863-867; D. Eyre et al., Ann. Rev. Biochem 53 (1984), 717-748 and D. Eyre, Methods in Enzymology 144 (1987), 115-139). These cross-links can be considered biochemical Marker for the specific detection of collagen can be used (Z. Gunja-Smith et al., Biochem. J. 197 (1981), 759-762). When extracellular collagen is broken down come hydroxylysylpyridinoline or lysylpyridinoline derivatives, which contain peptide side chains or free ones Pyridinoline derivatives with lysyl or hydroxylysyl residues, such as described in WO 91/10141, in body fluids such as Blood or urine. Evidence of these compounds in body fluids therefore gives indications of the breakdown of extracellular Collagen, e.g. in osteoporosis as well occurs as a result of tumors of the bone tissue. As proof of such hydroxylysyl or lysyl pyridinolines with peptide side chains were monoclonal antibodies in WO 89/12824 described, which by immunization with appropriate cross-linked collagen fragments were obtained from can be isolated from the urine. Also in the case of WO 91/08478 described method, the detection of collagen via an antibody against natural, i.e. generated in vivo cross-linked breakdown products of collagen.
Ein Nachteil dieser aus natürlichen Quellen isolierten Peptide ist, daß keine sichere Quelle für eine reproduzierbare Herstellung der Antigene oder Bindepartner im Test vorhanden ist. Ein weiterer Nachteil der aus natürlichen Quellen isolierten Peptide ist die Gefahr einer Kontamination mit infektiösem Material.A disadvantage of this isolated from natural sources Peptides is not a sure source of reproducible Production of the antigens or binding partners present in the test is. Another disadvantage of natural sources isolated peptides is at risk of contamination infectious material.
Definierte Antigene können z.B. durch chemische Synthese eines Peptids erhalten werden, welches einem Epitop des Antigens entspricht. Werden hierfür kleine Peptide mit einem Molekulargewicht von etwa 700 - 1500 D verwendet, so ist die Bindung an ein Trägermolekül erforderlich, um ein Antigen mit immunogener Wirkung zu erhalten. Durch die Bindung an das Trägermolekül darf dabei die Struktur des Epitops nicht verändert werden. Die Kupplung an das Trägermolekül erfolgt daher bislang bevorzugt an den Enden der Peptidkette in ausreichendem Abstand von dem vermuteten Epitopbereich (Laboratory Technics in Biochemistry and Molecular Biology, Synthetic Polypeptides as Antigens, Editors R.H. Burdon und P.H. van Knippenberg, Elsevier, Amsterdam, New York, Oxford 1988, Seiten 95-100).Defined antigens can e.g. through chemical synthesis of a peptide which is an epitope of the antigen corresponds. Are small peptides with a Molecular weight of about 700 - 1500 D used, that is Binding to a carrier molecule is required to use an antigen to obtain immunogenic effect. By binding to that The carrier molecule must not change the structure of the epitope will. The coupling to the carrier molecule therefore takes place previously preferred at the ends of the peptide chain in sufficient distance from the suspected epitope area (Laboratory Technics in Biochemistry and Molecular Biology, Synthetic Polypeptides as Antigens, Editors R.H. Burdon and P.H. van Knippenberg, Elsevier, Amsterdam, New York, Oxford 1988, pages 95-100).
Ein Problem bei der chemischen Synthese eines definierten Antigens, das einem natürlichen Abbauprodukt von quervernetztem Kollagen entspricht, stellt die aus der Quervernetzung resultierende Hydroxylysyl- oder LysylpyridinolinStruktur dar, deren chemische Synthese bislang nicht gelungen ist.A problem in the chemical synthesis of a defined Antigen, which is a natural degradation product of cross-linked Corresponds to collagen, which represents the cross-linking resulting hydroxylysyl or lysylpyridinoline structure whose chemical synthesis has so far not been successful is.
Aufgabe der Erfindung war es daher, ein definiertes Antigen zur Herstellung von Antikörpern gegen Kollagen Typ I oder Kollagenfragment davon, zum Einsatz als spezifischer Bindepartner des Antikörpers gegen Kollagen oder Kollagenfragmente in einem kompetitiven Immunoassay und als Standardmaterial zur Erstellung einer Standard- oder Eichkurve in einem kompetitiven Immunoassay zum Nachweis von Kollagen oder Kollagenfragmenten vom Typ I zur Verfügung zu stellen.The object of the invention was therefore to create a defined antigen for the production of antibodies against type I collagen or a collagen fragment of which, for use as a specific binding partner of Antibody to collagen or collagen fragments in one competitive immunoassay and as standard material for preparation a standard or calibration curve in a competitive Immunoassay for the detection of collagen or type I collagen fragments to provide.
Bisher wurde immer davon ausgegangen, daß es zum Nachweis der Kollagen oder Kollagenabbauprodukte in einer Probe notwendig sei, die Quervernetzungsstrukturen an sich oder sogenannte quervernetzte Peptide, die durch die Quervernetzung der Hydroxylysyl- oder Lysylreste zustandekommen, nachzuweisen, da diese Hydroxylysyl- oder Lysylpyridinolinstruktur charakteristisch für Kollagen ist. Beispiele für solche Nachweismethoden sind in der WO 89/12824, WO 91/08478, WO 89/04491 und der WO 91/10141 beschrieben. So far it has always been assumed that it is used to prove the Collagen or collagen breakdown products required in a sample be the cross-linking structures per se or so-called cross-linked peptides by cross-linking the Hydroxylysyl or lysyl residues because this hydroxylysyl or lysylpyridinoline structure is characteristic is for collagen. Examples of such detection methods are in WO 89/12824, WO 91/08478, WO 89/04491 and WO 91/10141.
Es wurde nun überraschenderweise gefunden, daß es zur Lösung der oben genannten Aufgabe ausreicht, ein definiertes Antigen, einen Bindepartner oder ein Standardmaterial zu verwenden, das ein synthetisches lineares Peptid enthält, das einer Sequenz des nichthelikalen linearen C- oder N-terminalen Bereiches von Kollagen Typ I entspricht. Die Vorteile bei Verwendung von synthetischen linearen Peptiden zur Verwendung als Bindepartner in Immunoassays, als Standardmaterial oder als Immunogen bei der Antikörper-Herstellung liegen darin, daß diese Peptide, im Gegensatz zu Peptiden aus natürlichen Quellen, in genau definierter Struktur reproduzierbar hergestellt werden können. Zudem zeigt ein Immunoassay, in dem solche kurzen synthetischen Peptide eingesetzt werden, eine geringere Störanfälligkeit.It has now surprisingly been found to be the solution the above task is sufficient, a defined antigen, to use a binding partner or a standard material, that contains a synthetic linear peptide that one Sequence of the non-helical linear C- or N-terminal Range of type I collagen. The benefits of using of synthetic linear peptides for use as Binding partner in immunoassays, as standard material or as Immunogen in antibody production is that these peptides, unlike natural peptides Sources, reproducibly produced in a precisely defined structure can be. In addition, an immunoassay shows in which such short synthetic peptides are used, a less susceptibility to failure.
Gegenstand der Erfindung ist daher, ein kompetitiver Immunoassay zum Nachweis von Kollagen Typ I oder Kollagenfragmenten in einer Probe, der dadurch gekennzeichnet ist, daß ein Bindepartner, der ein synthetisches lineares Peptid enthält, das einer Sequenz des nichthelikalen linearen C- oder N-terminalen Bereiches von Kollagen Typ I entspricht, mit einem Antikörper, der das synthetische lineare Peptid zu binden vermag, und der Probe inkubiert und die Bindung des Antikörpers an den Bindepartner in geeigneter Weise bestimmt wird.The object of the invention is therefore a competitive immunoassay for the detection of collagen type I or collagen fragments in a sample, which is characterized in that a binding partner, which contains a synthetic linear peptide which a sequence of the non-helical linear C- or N-terminal Region of type I collagen, with an antibody, which is able to bind the synthetic linear peptide, and the Incubated sample and binding of the antibody to the binding partner is determined in a suitable manner.
Ein weiterer Gegenstand der Erfindung ist ein Standardmaterial zur Erstellung einer Standard- oder Eichkurve in einem kompetitiven Immunoassay zum Nachweis von Kollagen oder Kollagenfragmenten vom Typ I, dadurch gekennzeichnet, daß ein Antigen enthalten ist, das ein synthetisches Peptid, das einer Sequenz des nichthelikalen C- oder N-terminalen Bereichs von Kollagen Typ I entspricht, enthält. Another object of the invention is a standard material to create a standard or calibration curve in a competitive immunoassay for the detection of collagen or Type I collagen fragments, characterized in that an antigen is contained, which is a synthetic peptide, which one Sequence of the non-helical C- or N-terminal region of Corresponds to type I collagen.
Ein weiterer Gegenstand der Erfindung ist ebenfalls ein Antigen zur Herstellung von Antikörpern gegen Kollagen Typ I oder Kollagenfragmente davon, das ein synthetisches lineares Peptid, das einer Sequenz des nichthelikalen C- oder N-terminalen Bereichs von Kollagen Typ I entspricht, enthält sowie die mit diesem Antigen hergestellten Antikörper.Another object of the invention is also an antigen for the production of antibodies against type I or collagen Collagen fragments thereof, which is a synthetic linear peptide, which a sequence of the non-helical C or N-terminal region of collagen type I contains, as well as contains with this Antigen produced antibodies.
Als synthetische lineare Peptide sind alle zusammenhängenden Aminosäuresequenzen des nichthelikalen C-oder N-terminalen Bereichs von Kollagen Typ I geeignet. Diese Bereiches sind aus Chu et al., Nature 310, 337-340 (1984), Click et al., Biochemistry 9, 4699-4706 (1970), Morgan et al., J. Biol. Chem. 245, 5042-5048 (1970) und Bernard et al., Biochemistry 22, 5213-5223 (1983) bekannt. Bevorzugt werden Peptide aus 5 bis 25 Aminosäuren, besonders bevorzugt aus 8 bis 20 Aminosäuren, verwendet. Dabei ist es nicht notwendig, daß die Sequenz den Bereich der Quervernetzung umfaßt. Sie kann aber sehr wohl ebenfalls mit diesem Bereich überlappen. In keinem Fall liegt aber in dem synthetischen Peptid eine Hydroxylysyl- oder Lysylpyridinolin-Quervernetzung vor. Am geeignetsten haben sich die synthetischen Peptide aus dem C-terminalen Bereich von Kollagen Typ I erwiesen, da der nichthelikale C-terminale Bereich größer als der nichthelikale N-terminale Bereich von Kollagen ist. In diesem Bereich stehen somit mehr potentielle Epitope als in den N-terminalen Bereich zur Verfügung. Peptide mit der in SEQ ID NO 1, 2 oder 3 gezeigten Sequenz aus dem C-terminalen Bereich der α1-Kette von Kollagen sind besonders geeignet.As synthetic linear peptides, all are related Amino acid sequences of the non-helical C- or N-terminal Range of type I collagen. These areas are out Chu et al., Nature 310, 337-340 (1984), Click et al., Biochemistry 9, 4699-4706 (1970), Morgan et al., J. Biol. Chem. 245: 5042-5048 (1970) and Bernard et al., Biochemistry 22, 5213-5223 (1983). Peptides from 5 are preferred up to 25 amino acids, particularly preferably from 8 to 20 amino acids, used. It is not necessary that the Sequence covers the area of cross-linking. But it can very well also overlap with this area. In none However, in the synthetic peptide there is a hydroxylysyl or lysylpyridinoline crosslinking. Most appropriate have the synthetic peptides from the C-terminal Area of type I collagen proven to be non-helical C-terminal area larger than the non-helical N-terminal Area of collagen. There are therefore more in this area potential epitopes are available as in the N-terminal area. Peptides with that shown in SEQ ID NO 1, 2 or 3 Sequence from the C-terminal region of the α1 chain of collagen are particularly suitable.
Die Konzentration von Abbauprodukten von Kollagen ist ein wichtiger diagnostischer Marker für das Ausmaß einer Osteolyse. Mit Hilfe der synthetischen linearen Peptide ist es möglich, einen kompetitiven Immunoassay zum Nachweis von Kollagen oder Kollagenfragmenten durchzuführen. Es hat sich überraschenderweise gezeigt, daß diese Peptide sehr gut mit den Kollagenfragmenten, die in natürlichen Proben wie Plasma, Serum oder Urin vorkommen, um Antikörper gegen diese Kollagenfragmente konkurrieren und damit einen kompetitiven Test ermöglichen. Solche Antikörper gegen Kollagen oder Kollagenabbauprodukte sind kommerziell erhältlich, beispielsweise in dem Telopeptide ICTP [125J) Radioimmunoassay Kit der Firma Orion Diagnostica, Finnland. Sie können aber ebenfalls erfindungsgemäß mittels des synthetischen linearen Peptids hergestellt werden. WO-A-90/08195 beschreibt einen Test zum Nachweis von Typ IX Kollagen, durch einen monoklonalen Antikörper, der mittels synthetischer Peptide, die Sequenzen der globulären Domäne NC 4 darstellen, hergestellt wurde. Die Verhältnisse bei Kollagen IX ist nicht mit Kollagen Typ I zu vergleichen; insbesondere tritt in der globularen Domäne bei Typ IX Kollagen keine charakteristische Quervernetzung auf. In EP-A-0298210 wird zum Immunisieren von Tieren eine Peptidsequenz verwendet, um Antikörper zu gewinnen, die Prokollagen vom Typ III erkennen. Prokollagen stellt keinen diagnostischen Marker für die Osteölyse dar.The concentration of collagen breakdown products is an important diagnostic marker for the extent of osteolysis. With the help of the synthetic linear peptides it is possible to carry out a competitive immunoassay for the detection of collagen or collagen fragments. It has surprisingly been found that these peptides compete very well with the collagen fragments that occur in natural samples such as plasma, serum or urine for antibodies against these collagen fragments and thus enable a competitive test. Such antibodies against collagen or collagen breakdown products are commercially available, for example in the telopeptide ICTP [ 125 I) radioimmunoassay kit from Orion Diagnostica, Finland. However, they can also be produced according to the invention using the synthetic linear peptide. WO-A-90/08195 describes a test for the detection of type IX collagen, by a monoclonal antibody, which was produced by means of synthetic peptides which represent sequences of the globular domain NC 4. The situation in collagen IX cannot be compared with type I collagen; in particular, no characteristic cross-linking occurs in the globular domain in type IX collagen. In EP-A-0298210 a peptide sequence is used to immunize animals in order to raise antibodies which recognize type III procollagen. Procollagen is not a diagnostic marker for osteolysis.
Zum Einsatz in einem kompetitiven Immunoassay kann das synthetische lineare Peptid direkt als Bindepartner, der an eine Festphase gebunden ist, benutzt werden oder aber gekoppelt an eine zweite Komponente. Die Kopplung an die zweite Komponente erfolgt bevorzugt über die N- und C-terminale Aminosäuren des linearen Peptids. Gegebenenfalls kann zwischen dem Peptid und der zweiten Komponente noch ein Spacer eingefügt werden. Die zweite Komponente kann beispielsweise dazu dienen, das Peptid indirekt an eine Festphase zu koppeln. Beispiele hierfür sind dem Fachmann bekannt. Bevorzugt wird das Peptid an Rinderserumalbumin gekoppelt und das Kopplungsprodukt adsorptiv an eine Festphase zum Beispiel ein Kunststoffröhrchen gebunden. Das Peptid kann auch an Biotin kovalent gebunden werden. Die Anheftung an die Festphase erfolgt dann über die Bindung an Avidin oder Streptavidin, das seinerseits an die Festphase gebunden wurde. Die zweite Komponente kann auch als Träger für mehrerer Peptide dienen, zum Beispiel in einem kompetitiven turbidimetrischen Inhibierung-Immunoassay (TINIA), wobei mehrere Peptide an beispielsweise Albumin, Immunglobuline, β-Galaktosidase, Polymere wie Polylysin oder Dextranmoleküle wie in der EP-A-0 545 350 beschrieben oder Partikel wie Latex gekoppelt sind. Vorzugsweise werden 30 bis 40 Peptidmoleküle je Trägermolekül gekoppelt. Das Peptid kann auch an eine Komponente, die eine Markierung darstellt, gekoppelt sein. Beispiele für a11 diese Testvarianten sind dem Fachmann bekannt.For use in a competitive immunoassay, the synthetic linear peptide directly as a binding partner that connects to a Solid phase is bound, used or coupled to a second component. The coupling to the second component is preferably carried out via the N- and C-terminal amino acids of the linear peptide. Optionally, between the peptide and a spacer can be inserted into the second component. The The second component can serve, for example, the peptide to couple indirectly to a solid phase. examples for this are known to the expert. The peptide is preferred on bovine serum albumin coupled and the coupling product adsorptively a solid phase, for example a plastic tube. The peptide can also be covalently bound to biotin. The Attachment to the solid phase then takes place via the bond to Avidin or streptavidin, which in turn adhere to the solid phase was bound. The second component can also be used as a carrier serve for multiple peptides, for example in a competitive turbidimetric inhibition immunoassay (TINIA), where several peptides on for example albumin, immunoglobulins, β-galactosidase, polymers such as polylysine or dextran molecules as described in EP-A-0 545 350 or particles how latex are coupled. Preferably 30 to 40 Peptide molecules coupled per carrier molecule. The peptide can also coupled to a component that represents a marker be. Examples of a11 these test variants are the Known specialist.
Bei der Test führung kann der Antikörper gleichzeitig oder sequentiell mit der Probe sowie dem Bindepartner, der das synthetische lineare Peptid enthält, inkubiert werden. Anschließend wird in üblicher Weise die Menge des gebundenen oder nichtgebundenen Antikörpers bestimmt. Als kompetitive Testvarianten zur Bestimmung der Menge an gebundenem oder ungebundenem Antikörper können beispielsweise Agglutinationstests, wie TINIA, oder FPIA- (Fluoreszenz-Polarisations-Immunoassay) (W. Dandliker et al., J.Exp. Med. 122 (1965), 1029), EMIT- (Enzyme Multiplied Immunoassay) (Gunzer et al., Kontakte III (1980), 3 - 11) und CEDIA-Prinzipien (Henderson et al., Clinical Chemistry 32 (1986), 1637 - 1641) dienen. Die erfindungsgemäßen Peptide haben sich als besonders geeignet für die Verwendung als definierte Bindepartner zur Kompetition mit der Probe um die Bindung an die Antikörper erwiesen. Besonders bevorzugt sind die synthetischen linearen Peptid mit der in SEQ ID NO 1, 2 oder 3 gezeigten Sequenz.The antibody can be used simultaneously or during the test sequentially with the sample and the binding partner who Contains synthetic linear peptide to be incubated. Subsequently is the amount of bound in the usual way or unbound antibody. As a competitive Test variants for determining the amount of bound or unbound antibody can, for example, agglutination tests, such as TINIA, or FPIA (fluorescence polarization immunoassay) (W. Dandliker et al., J.Exp. Med. 122 (1965), 1029), EMIT (Enzyme Multiplied Immunoassay) (Gunzer et al., Contacts III (1980), 3 - 11) and CEDIA principles (Henderson et al., Clinical Chemistry 32 (1986), 1637-1641). The peptides according to the invention have proven to be special suitable for use as a defined binding partner for Competition with the sample for binding to the antibodies proven. The synthetic linear ones are particularly preferred Peptide with the sequence shown in SEQ ID NO 1, 2 or 3.
Nach der Bestimmung des Ausmaßes der Bindung des Antikörpers an den Bindepartner, das ein Maß für die Menge an Antigen in der Probe darstellt, kann die genaue Menge an Antigen in der Probe in üblicher Weise durch Vergleich mit in gleicher Weise behandeltem Standard ermittelt werden.After determining the extent of antibody binding to the binding partner, which is a measure of the amount of antigen in represents the exact amount of antigen in the sample Sample in the usual way by comparing with in the same way treated standard can be determined.
Als Standard können Kollagenabbauprodukte isoliert aus natürlichem Material verwendet werden. Diese zeichnen sich jedoch naturgemäß durch eine bestimmte Schwankung aus. Geeigneter erwiesen hat sich als Standardmaterial ein Antigen, das das erfindungsgemäße synthetische lineare Peptid enthält. Das Antigen des Standards kann dabei lediglich aus diesem Peptid bestehen oder aber aus diesem Peptid, das an einen geeigneten Träger gekoppelt ist, der beispielsweise zur besseren Wasserlöslichkeit des Peptids dient. Zur Herstellung des Standard-materials aus Peptid und Träger wird das lineare Peptid, das einer Sequenz des nichthelikalen C- oder N-terminalen Bereichs von Kollagen Typ I entspricht, synthetisiert und über seine N- oder C-terminale Aminosäure durch geeignete Kopplungs-methoden an das Trägermolekül gebunden. Es können ein oder mehrere Peptide pro Trägermolekül gebunden werden. Gegebenenfalls kann die Kopplung über einen Spacer erfolgen. Für bestimmte Zwecke, wie beispielsweise für Agglutinationstests, kann es von Vorteil sein, mehrere erfindungsgemäße Peptide unterschiedlicher Sequenz an ein Trägermolekül zu binden, vor allem falls polyklonale Antikörper im Test eingesetzt werden, die nicht mit Hilfe des erfindungsgemäßen Antigens hergestellt wurden und damit meist mehrere Epitope erkennen.As a standard, collagen breakdown products can be isolated from natural Material can be used. However, these stand out naturally by a certain fluctuation. More suitable An antigen that has proven to be the standard material contains synthetic linear peptide according to the invention. The Antigen of the standard can only be made from this peptide consist of or from this peptide attached to a suitable Carrier is coupled, for example, for better water solubility of the peptide. For the production of the standard material the peptide and carrier become the linear peptide, the a sequence of the non-helical C or N-terminal region of type I collagen, synthesized and via its N- or C-terminal amino acid using suitable coupling methods bound to the carrier molecule. It can be a or several peptides are bound per carrier molecule. Possibly the coupling can take place via a spacer. For certain Purposes, such as for agglutination tests, it may be advantageous to use several peptides according to the invention different sequence to bind to a carrier molecule especially if polyclonal antibodies are used in the test, which are not produced with the aid of the antigen according to the invention and usually recognize several epitopes.
Als Antikörper in dem kompetitiven Immunoassay können die bereits bekannten Antikörper gegen Kollagenabbauprodukte verwendet werden. Geeignet sind auch Antikörper, die mit Hilfe eines Antigens, das das erfindungsgemäße lineare synthetische Peptid enthält, gewonnen wurden.They can already be used as antibodies in the competitive immunoassay known antibodies against collagen breakdown products used will. Antibodies with the help are also suitable of an antigen which is the linear synthetic according to the invention Contains peptide were obtained.
Zur Immunisierung müssen die linearen synthetischen Peptide, die einer oder mehrerer Sequenzen des nichthelikalen C- oder N-terminalen Bereichs von Kollagen Typ I entsprechen, an ein geeignetes Trägerprotein, wie zum Beispiel Keyhole Limpet Hemocyanine, Rinderserumalbumin oder Edestin gebunden werden. Zur Herstellung dieser Antigene oder Immunogene werden zunächst die linearen Peptide in üblicher Weise chemisch synthetisiert. Anschließend erfolgt die Kopplung der synthetischen Peptide über die N-terminale Aminogruppe mittels Maleinimidohexansäure-N-hydroxysucciminidester an die oben genannten Trägerproteine. Es hat sich überraschenderweise gezeigt, daß zur Herstellung von Antikörpern, die für die kompetitive Testführung geeignet sind, synthetische lineare Peptide mit der in SEQ ID NO 1, 2 oder 3 gezeigten Sequenz besonders geeignet sind.For immunization, the linear synthetic peptides, that of one or more sequences of the non-helical C- or N-terminal region of collagen type I correspond to a suitable one Carrier protein such as keyhole limpet hemocyanins, Bovine serum albumin or edestin can be bound. To produce these antigens or immunogens are first the linear peptides chemically in the usual way synthesized. The synthetic is then coupled Peptides via the N-terminal amino group Maleinimidohexanoic acid N-hydroxysucciminide ester to the above called carrier proteins. It has surprisingly turned out to be shown that for the production of antibodies for the competitive test management are suitable, synthetic linear Peptides with the sequence shown in SEQ ID NO 1, 2 or 3 are particularly suitable.
Mit den erfindungsgemäßen Antigenen, die ein synthetisches lineares Peptid enthalten, das einer Sequenz des nichthelikalen C- oder N-terminalen Bereichs von Kollagen Typ I entspricht, ist es möglich, Antikörper zu erhalten, welche nicht nur das erfindungsgemäße Peptid sondern auch die in Körperflüssigkeiten vorkommenden Abbauprodukte von Kollagen erkennen.With the antigens according to the invention, which is a synthetic linear peptide containing a sequence of the non-helical Corresponds to the C- or N-terminal region of collagen type I, it is possible to get antibodies that do not only the peptide according to the invention but also those in body fluids Detect occurring breakdown products of collagen.
Ein weiterer Gegenstand der Erfindung ist daher ein Verfahren zur Herstellung von Antikörpern gegen Kollagen oder Kollagenfragmente durch Immunisierung mit einem erfindungsgemäßen Antigen und Isolierung des gewünschten Antikörpers aus dem Serum der immunisierten Tiere nach bekannten Verfahren. Vorzugsweise erfolgt die Isolierung des gewünschten Antikörpers über Immunadsorption an einem an ein Trägerprotein, vorzugsweise Sepharose, gekoppelten Peptid der in SEQ ID NO 1, 2 oder 3 gezeigten Sequenz.Another object of the invention is therefore a method for the production of antibodies against collagen or collagen fragments by immunization with an inventive Antigen and isolation of the desired antibody from the Serum of the immunized animals according to known methods. Preferably the desired antibody is isolated via immunoadsorption on a carrier protein, preferably Sepharose, coupled peptide of the SEQ ID NO 1, 2 or 3 sequence shown.
Ein bevorzugter Gegenstand der Erfindung ist ein Verfahren zur Herstellung monoklonaler Antikörper gegen Kollagen oder Kollagenfragmente durch Immunisierung mit einem erfindungsgemäßen Antigen, Immortalisierung der Milzzellen der immunisierten Tiere, Klonierung derjenigen immortalisierten Milzzellen, welche den gewünschten Antikörper produzieren und Isolierung des Antikörpers aus den klonierten Zellen oder dem Kulturüberstand dieser Zellen.A preferred subject of the invention is a method for the production of monoclonal antibodies against collagen or Collagen fragments by immunization with an inventive Antigen, immortalization of the spleen cells of the immunized Animals, cloning of those immortalized spleen cells, which produce the desired antibody and Isolation of the antibody from the cloned cells or the Culture supernatant of these cells.
Die Immunisierung erfolgt in den hierfür üblicherweise verwendeten Tieren, vorzugsweise werden Mäuse oder Kaninchen verwendet.The immunization takes place in the commonly used for this Animals, preferably mice or rabbits used.
Die Immortalisierung der Milzzellen der immunisierten Tiere erfolgt nach dem Fachmann geläufigen Verfahren, wie z.B. der Hybridomtechnik (Köhler und Milstein, Nature 256 (1975), 495 - 497) oder durch Transformation mit dem Epstein-Barr-Virus (EBV-Transformation). Zum Nachweis derjenigen immortalisierten Zellen, die den gewünschten Antikörper produzieren, wird eine Probe des Kulturüberstands in einem üblichen Immunoassay mit dem zur Immunisierung verwendeten erfindungsgemäßen Antigen inkubiert und untersucht, ob ein Antikörper an dieses Antigen bindet.Immortalization of the spleen cells of the immunized animals takes place according to methods familiar to the person skilled in the art, e.g. of the Hybridoma technology (Köhler and Milstein, Nature 256 (1975), 495 - 497) or by transformation with the Epstein-Barr virus (EBV transformation). To prove those immortalized Cells that produce the desired antibody a sample of the culture supernatant in a conventional immunoassay with the antigen according to the invention used for immunization incubated and examined for an antibody to this Antigen binds.
Ein weiterer Gegenstand der Erfindung sind die nach dem erfindungsgemäßen Verfahren erhältlichen polyklonalen und monoklonalen Antikörper.Another object of the invention are those according to the invention Polyclonal and monoclonal methods available Antibody.
Diese polyklonalen und monoklonalen Antikörper reagieren nicht nur mit dem zur Immunisierung verwendeten erfindungsgemäßen Hapten, sondern gut mit Kollagen, sowie mit den in Körperflüssigkeiten gefundenen natürlichen Abbauprodukten von Kollagen.These polyclonal and monoclonal antibodies react not only with the immunization according to the invention used Hapten, but good with collagen, as well as with the in Body fluids found natural breakdown products of Collagen.
Die erfindungsgemäßen Antikörper können daher in den oben beschriebenen Nachweisverfahren zur Bestimmung von Kollagen oder Kollagenfragmenten verwendet werden. The antibodies of the invention can therefore in the above described detection methods for the determination of collagen or collagen fragments can be used.
Ein weiterer Gegenstand der Erfindung ist daher die Verwendung eines erfindungsgemäßen polyklonalen oder monoklonalen Antikörpers zur Bestimmung der Osteolyse durch Inkubation des Antikörpers mit einer Gewebeprobe und Bestimmung des an den Antikörper bindenden Kollagenabbauproduktes.Another object of the invention is therefore the use of a polyclonal or monoclonal according to the invention Antibody for the determination of osteolysis by incubation of the Antibody with a tissue sample and determination of the to the Antibody binding collagen breakdown product.
Die Erfindung wird durch die folgenden Beispiele in Verbindung mit den Sequenzprotokollen näher erläutert.
- SEQ ID NO 1
- zeigt die Sequenz eines erfindungsgemäßen Peptids aus 9 Aminosäuren, wobei Xaa eine beliebige Aminosäure bedeutet.
- SEQ ID NO 2
- zeigt die Sequenz eines erfindungsgemäßen Peptids aus 16 Aminosäuren.
- SEQ ID NO 3
- zeigt die Sequenz eines erfindungsgemäßen Peptids aus 10 Aminosäuren.
- SEQ ID NO 1
- shows the sequence of a peptide according to the invention from 9 amino acids, where Xaa means any amino acid.
- SEQ ID NO 2
- shows the sequence of a peptide of 16 amino acids according to the invention.
- SEQ ID NO 3
- shows the sequence of a peptide of 10 amino acids according to the invention.
Die eine Teilsequenz der Aminosäurensequenz von Kollagen
aufweisenden Peptide der in den Sequenzprotokollen SEQ ID
NO 2 und 3 gezeigten Sequenzen werden mittels Fluorenylmethyloxycarbonyl(Fmoc)-Festphasenpeptidsynthese
an einem a)
Labortec SP 640 Peptide Synthesizer bzw. b) Zinsser Analytic
SMPS 350 Peptidsynthesizer hergestellt.
- Lys
- mit tert. Butyloxycarbonyl-Schutzgruppe
- Glu
- mit tert. Butylester-Schutzgruppe
- Gln
- ohne Seitenketten-Schutzgruppe
- Pro
- ohne Seitenketten-Schutzgruppe
- Pro
- ohne Seitenketten-Schutzgruppe
- Gln
- ohne Seitenketten-Schutzgruppe
- Pro
- ohne Seitenketten-Schutzgruppe
- Leu
- ohne Seitenketten-Schutzgruppe
- Phe
- ohne Seitenketten-Schutzgruppe
- Ser
- mit tert. Butylether-Schutzgruppe
- Phe
- ohne Seitenketten-Schutzgruppe
- Asp
- mit tert. Butylester-Schutzgruppe
- Phe
- ohne Seitenketten-Schutzgruppe
- Gly
- ohne Seitenketten-Schutzgruppe
- Ala
- ohne Seitenketten-Schutzgruppe
- Ser
- mit tert. Butylether-Schutzgruppe
- Acetyl
- Essigsäureanhydrid
- Glu
- mit Trityl-Schutzgruppe
- Ser
- mit tert. Butyl-Schutzgruppe
- Asp
- mit tert. Butyl-Schutzgruppe
- Lys
- with tert. Butyloxycarbonyl protecting group
- Glu
- with tert. Butyl ester protecting group
- Gln
- without side chain protection group
- Per
- without side chain protection group
- Per
- without side chain protection group
- Gln
- without side chain protection group
- Per
- without side chain protection group
- Leu
- without side chain protection group
- Phe
- without side chain protection group
- Ser
- with tert. Butyl ether protecting group
- Phe
- without side chain protection group
- Asp
- with tert. Butyl ester protecting group
- Phe
- without side chain protection group
- Gly
- without side chain protection group
- Ala
- without side chain protection group
- Ser
- with tert. Butyl ether protecting group
- Acetyl
- Acetic anhydride
- Glu
- with trityl protecting group
- Ser
- with tert. Butyl protecting group
- Asp
- with tert. Butyl protecting group
Die Kupplungszeiten betrugen 30 und 40 Minuten. Die Abspaltzeit betrug 20 Minuten und wurde mit einer Lösung von 50% Piperidin im DMF durchgeführt. Die Waschschritte wurden mit DMF achtmal nach jedem Reaktionsschritt durchgeführt. Die Freisetzung des Peptids erfolgte durch Behandlung des vom Lösungsmittel abfiltrierten und mit Dichlormethan und Methanol gewaschenen Harz mit 1 ml einer Lösung von 90% Trifluoressigsäure, 3% Thioanisol, 3 % Ethandithiol und 3 % Thiokresol innerhalb von 20 Minuten und 140 Minuten. Das Produkt wurde durch Zugabe vom 15 ml kaltem Diisopropylether zum vereinigten Filtrat ausgefällt und durch Filtration isoliert. Der Rückstand wurde in 50%iger Essigsäure gelöst und lyophilisiert. Es wurden 8 mg weißes Lyophilisat einer Reinheit von 79% laut HPLC erhalten. Die Identität wurde mittels FAB-Massenspektroskopie bestätigt.The coupling times were 30 and 40 minutes. The split time was 20 minutes and was with a 50% solution Piperidine performed in the DMF. The washing steps were with DMF performed eight times after each reaction step. The The peptide was released by treatment of the Filtered off solvent and with dichloromethane and methanol washed resin with 1 ml of a solution of 90% trifluoroacetic acid, 3% thioanisole, 3% ethanedithiol and 3% thiocresol within 20 minutes and 140 minutes. The product was combined by adding 15 ml of cold diisopropyl ether Filtrate precipitated and isolated by filtration. The residue was dissolved in 50% acetic acid and lyophilized. 8 mg of white lyophilizate were of a purity obtained by 79% according to HPLC. The identity was determined using FAB mass spectroscopy approved.
Das gemäß Beispiel la) hergestellte Peptid wird durch Acylierung mit Maleinimidohexanoyl-N-hydroxysuccinimid (MHS) aktiviert. Dazu wird 0,1 mmol des Peptids in 20 ml 0,1 mol/l Kaliumphosphatpuffer pH 7,5 gelöst, mit einer Lösung von 0,1 mmol MHS in 6 ml Dioxan versetzt und 20 min. bei 20°C gerührt. Anschließend wird der pH-Wert mit Eisessig auf pH 4 eingestellt und das Reaktionsgemisch sofort lyophilisiert. Das Lyophilisat wird in 5 ml Wasser gelöst und über präparative HPLC an einer Waters Delta-Pak® C18-Säule (100 Å, 15 µm 50 x 300 mm) über einen Elutionsgradienten von 100 % A (Wasser 0,1 % Trifluoressigsäure) zu 100 % B (99,9 % Acetonitril 0,1 % Trifluoressigsäure) aufgereinigt. The peptide produced according to example la) is by acylation with maleimidohexanoyl-N-hydroxysuccinimide (MHS) activated. For this, 0.1 mmol of the peptide in 20 ml becomes 0.1 mol / l Potassium phosphate buffer pH 7.5 dissolved, with a solution of 0.1 mmol of MHS in 6 ml of dioxane and 20 min. stirred at 20 ° C. Then the pH is adjusted to pH 4 with glacial acetic acid adjusted and the reaction mixture immediately lyophilized. The lyophilisate is dissolved in 5 ml of water and preparative HPLC on a Waters Delta-Pak® C18 column (100 Å, 15 µm 50 x 300 mm) over an elution gradient of 100% A. (Water 0.1% trifluoroacetic acid) to 100% B (99.9% acetonitrile 0.1% trifluoroacetic acid).
Beschrieben wird die Kopplung von aktivierten Peptiden an Keyhole Limpet Hemocyanine (KLH), Rinderserumalbumin (RSA) und β-Galaktosidase (βGal). Zur Kopplung der nach Beispiel 2 mit MHS aktivierten Peptide ist es erforderlich, daß das Trägerprotein freie SH-Gruppen trägt. βGal weist diese natürlicherweise bereits auf, erfordert also keine weitere Vorbehandlung. Bei KLH und RSA werden die NH2-Gruppen der ε-Aminoseitenkette von Lysinresten durch Behandlung mit N-Succinimidyl-S-acetylthiopropionat (SATP) deriatisiert und dadurch in SH-Gruppen umgewandelt.The coupling of activated peptides to keyhole limpet hemocyanins (KLH), bovine serum albumin (RSA) and β-galactosidase (βGal) is described. To couple the peptides activated with MHS according to Example 2, it is necessary that the carrier protein carries free SH groups. βGal naturally already has this, so it does not require any further pretreatment. In KLH and RSA, the NH 2 groups of the ε-amino side chain of lysine residues are deriatized by treatment with N-succinimidyl-S-acetylthiopropionate (SATP) and thereby converted into SH groups.
Man erhält somit ein Trägerprotein das eine gegenüber dem nativen Zustand erhöhte Anzahl von SH-Gruppen aufweist. Hierfür wird zu einer Lösung aus 1,39 g KLH in 500 ml 0,1 mol/l Kaliumphosphatpuffer pH 8,5 innerhalb von 20 min. 113,51 mg SATP (in 10 ml Dioxan gelöst) zugetropft. Nach 30 min. Rühren bei 20°C wird der pH-Wert der Reaktionslösung mit 0,1 mol/l Natronlauge auf pH 8,5 nachgestellt und weitere 24 Stunden gerührt. Die Lösung wird anschließend mit Hilfe einer Amiconzelle (Membran YM10) auf 100 ml aufkonzentriert, 3 x 24 Stunden gegen je 3 1 0,1 mol/l Kaliumphosphatpuffer pH 8,5/ 0,05 mol/l Natriumchlorid dialysiert und anschließend lyophilisiert. A carrier protein is thus obtained, one versus the other native state has increased number of SH groups. Therefor becomes a solution of 1.39 g KLH in 500 ml 0.1 mol / l Potassium phosphate buffer pH 8.5 within 20 min. 113.51 mg SATP (dissolved in 10 ml of dioxane) was added dropwise. After 30 min. stir at 20 ° C the pH of the reaction solution becomes 0.1 mol / l Sodium hydroxide solution adjusted to pH 8.5 and another 24 hours touched. The solution is then made using an Amicon cell (Membrane YM10) concentrated to 100 ml, 3 x 24 Hours against 3 1 0.1 mol / l potassium phosphate buffer pH 8.5 / 0.05 mol / l sodium chloride dialyzed and then lyophilized.
Zur Abspaltung der S-Acetylschutzgruppe werden 481 mg des KLH-SATP Lyophilisats in 20 ml 0,1 mol/l Kaliumphosphatpuffer pH 8,5/0,05 mol/l Natriumchlorid gelöst, mit 0,5 ml frisch zubereiteter 1 mol/l Hydroxylaminlösung versetzt und 90 min. bei 20°C gerührt.To split off the S-acetyl protective group, 481 mg of the KLH-SATP lyophilisate in 20 ml 0.1 mol / l potassium phosphate buffer pH 8.5 / 0.05 mol / l sodium chloride dissolved, with 0.5 ml fresh prepared 1 mol / l hydroxylamine solution and 90 min. stirred at 20 ° C.
7,23 mol des aus Beispiel 2 erhaltenen aktivierten Peptids in 4 ml Wasser werden zu dem derivatisierten Trägerprotein gegeben und 20 Stunden bei 20°C gerührt. Anschließend wird die trübe Lösung 2 x gegen 1 1 0,1 mol/l Kaliumphosphatpuffer pH 8,5/0,05 mol/l Natriumchlorid dialysiert. Das Dialysat wird zentrifugiert, der klare Überstand abdekantiert und lyophilisiert.7.23 mol of the activated peptide obtained from Example 2 in 4 ml of water are added to the derivatized carrier protein and stirred at 20 ° C for 20 hours. Then the cloudy solution 2 x against 1 1 0.1 mol / l potassium phosphate buffer pH 8.5 / 0.05 mol / l sodium chloride dialyzed. The dialysate will centrifuged, the clear supernatant decanted and lyophilized.
Je 5 Schafe wurden in an sich bekannter Weise mit dem Immunogen aus Beispiel 3 immunisiert. Die Immunogene enthielten das Peptid mit der in SEQ ID NO 2 genannten Sequenz, das den Aminosäuren Nr. 892 bis 907 in der Sequenz der α-Kette von Kollagen Typ I entspricht. Als Trägerprotein diente KLH bzw. β-Galaktosidase. Die Tiere wurden in monatlichen Abständen mit den Immunogenen in kompletten Freundschen Adjuvans immunisiert. Die Dosis betrug 500 µg je Tier und Immunisierung. Vier Monate nach Erstimmunisierung wurden Blutproben entnommen und die erhaltenen Antikörper auf Reaktion mit Kollagenfragmenten geprüft. 5 sheep each were treated with the immunogen in a manner known per se immunized from Example 3. The immunogens contained that Peptide with the sequence mentioned in SEQ ID NO 2, which the Amino acids Nos. 892 to 907 in the sequence of the α chain of Corresponds to type I collagen. KLH or β-galactosidase. The animals were monthly with the immunogens in complete Freund's adjuvant immunized. The dose was 500 µg per animal and immunization. Blood samples were taken four months after first immunization removed and the antibodies obtained in response to Collagen fragments checked.
Folgendes Material und Reagenzien wurden verwendet:
Die Vertiefungen der Titerplatten wurden mit je 100 µl einer Lösung gefüllt, die 10 µg/ml Kollagenfragmente in Beschichtungspuffer enthielt. Die Kollagenfragmente wurden entsprechend den Angaben in EP-A-0 505 210 durch Protease-Verdauung von humanem Kollagen aus Knochen hergestellt. Nach einer Stunde Inkubation bei Raumtemperatur unter Schütteln wurde dreimal mit Waschlösung gewaschen. The wells of the titer plates were each 100 µl Solution filled, the 10 µg / ml collagen fragments in coating buffer contained. The collagen fragments were made accordingly the information in EP-A-0 505 210 by protease digestion made from human collagen from bones. After one hour incubation at room temperature with shaking was washed three times with washing solution.
Die Antiseren wurden mit Inkubationspuffer 1 : 4000 verdünnt und je 100 µl in den Vertiefungen der Mikrotiterplatte eine Stunde unter Schütteln bei Raumtemperatur inkubiert. Anschließend wurden die Vertiefungen dreimal mit Waschlösung gewaschen.The antisera were diluted 1: 4000 with incubation buffer and 100 µl each in the wells of the microtiter plate Incubated for one hour with shaking at room temperature. Subsequently the wells were washed three times with wash solution washed.
Ein Konjugat aus Meerettich-Peroxidase mit Kaninchen-Antikörpern gegen den Fc-Teil von Schaf-IgG wird im Inkubationspuffer bis zur Konzentration von 12,5 mU/ml verdünnt und die Näpfe der Mikrotiterplatte mit je 100 µl davon beschickt. Nach einer Stunde Inkubation unter Schütteln bei Raumtemperatur werden die Titerplatten dreimal mit Waschlösung gewaschen.A conjugate of horseradish peroxidase with rabbit antibodies against the Fc part of sheep IgG is in the incubation buffer diluted to a concentration of 12.5 mU / ml and the Wells of the microtiter plate are loaded with 100 µl each. After an hour of incubation with shaking at room temperature the titer plates are washed three times with washing solution.
100 µl Substratlösung werden hinzugefügt und inkubiert, bis eine Farbentwicklung deutlich wird (10 - 60 Minuten). Die Extinktion wird als Differenzmessung bei 405 und 492 nm erfaßt.100 µl substrate solution are added and incubated until a color development becomes clear (10 - 60 minutes). The Absorbance is recorded as a difference measurement at 405 and 492 nm.
Die Seren der meisten Tiere zeigten eine starke Reaktion mit
den Kollagenfragmenten auf der Festphase. Das Serum eines
nicht immunisierten Tieres zeigte unter gleichen Bedingungen
nur ein schwaches Meßsignal. Die Ergebnisse sind in der
Tabelle 1 dargestellt.
Die Vertiefungen einer 96-er Mikrotiterplatte werden gemäß
EP-A 0 344 578 mit Streptavidin (100 µl einer Lösung von
1 µg/ml in PBS) bei 4°C über Nacht beschichtet und noch freie
unspezifische Bindungsstellen durch Inkubation mit 300µl BSA
(Rinderserumalbumin, 10 mg/ml) für 2 Stunden bei Raumtemperatur
blockiert. The wells of a 96-well microtiter plate are made according to
EP-
Das Decapeptid mit der in SEQ ID NO 3 gezeigten Sequenz, das
gemäß Beispiel 1b) hergestellt wurde, wird aminoterminal mit
D-Biotinyl-ε-amidocapronsäure-N-succinimidester (Boehringer
Mannheim, Katalog Nr. 1008960) gemäß Angaben des Herstellers
biotinyliert. Das biotinylierte Peptid wird in PBS, 0,05%
Tween 20, 1% BSA in einer Konzentration von 10ng/ml gelöst
und durch 1-stündige Inkubation von 100µl je Vertiefung an
die mit Streptavidin beschichtete Mikrotiterplatte gebunden.
Anschließend wird ungebundenes Peptid durch dreimaliges
Waschen mit PBS, 0,05% Tween 20 entfernt.The decapeptide with the sequence shown in SEQ ID NO 3, the
was prepared according to Example 1b), with amino terminal
D-Biotinyl-ε-amidocaproic acid-N-succinimide ester (Boehringer
Mannheim, catalog No. 1008960) according to the manufacturer's instructions
biotinylated. The biotinylated peptide is in PBS, 0.05
Jeweils 150 µl der zu untersuchenden Probe (Serum, Plasma oder ein Standard) werden mit 150 µl des erfindungsgemäßen Antikörpers nach Beispiel 4 für 2 Stunden bei 37°C (oder über Nacht bei 4°C) inkubiert. Jeweils 100µl dieses Ansatzes werden in die Vertiefungen der Mikrotiterplatte zu dem gebundenen Decapeptid gegeben und 60 Minuten bei 37°C inkubiert. Dabei kann nur der nach der Inkubation mit der Probe noch nicht gebundene Antikörperüberschuß des Antiserums an das immobilisierte Decapeptid binden.150 µl each of the sample to be examined (serum, plasma or a standard) with 150 ul of the invention Antibody according to Example 4 for 2 hours at 37 ° C (or above Incubated at 4 ° C). 100µl of this approach each are bound into the wells of the microtiter plate Decapeptide added and incubated at 37 ° C for 60 minutes. Only the one after incubation with the sample can unbound antibody excess of the antiserum bind the immobilized decapeptide.
Nach dreimaligem Waschen mit PBS/0,05% Tween 20 wird gebundener
Antikörper durch anschließende Inkubation mit einem
Anti-Kaninchen-IgG-POD Konjugat (Boehringer Mannheim GmbH,
Katalog Nr. 1238 850) und ABTS® (1 mg/ml) nachgewiesen.After washing three times with PBS / 0.05
Mit dem erfindungsgemäßen Test (MTP Kompetitiver Test) wurden 164 Patienten-Seren vermessen. Die Ergebnisse wurden zu Daten, welche mit einem Radioimmunoassay (RIA) ermittelt wurden, in Bezug gesetzt. Dieser RIA ICTP (Telopeptide ICTP [125J] von Orion Diagnostica, Finnland) basiert auf quervernetzten Kollagenfragmenten, welche durch enzymatischen Verdau und biochemische Verfahren hergestellt und isoliert werden. Aus Figur 1 geht nun hervor, daß das erfindungsgemäße Verfahren Meßwerte erbringt, die gut mit den RIA-Werten korrelieren, das heißt, das erfindungsgemäße Verfahren liefert klinisch relevante Daten. Es wurde ein Korrelationskoeffizient von 0,959 ermittelt. 164 patient sera were measured with the test according to the invention (MTP competitive test). The results were related to data obtained using a radioimmunoassay (RIA). This RIA ICTP (Telopeptide ICTP [ 125 J] from Orion Diagnostica, Finland) is based on cross-linked collagen fragments, which are produced and isolated by enzymatic digestion and biochemical processes. It now appears from FIG. 1 that the method according to the invention produces measured values which correlate well with the RIA values, that is to say the method according to the invention provides clinically relevant data. A correlation coefficient of 0.995 was determined.
Claims (13)
- Competitive immunoassay for the detection of collagen type I or collagen fragments thereof in a sample, wherein a binding partner which contains a synthetic linear peptide that corresponds to a sequence of the non-helical C- or N-terminal region of collagen type Iis incubated with an antibody that is capable of binding the synthetic linear peptide,and the sampleand the binding of the antibody to the binding partner is determined in a suitable manner.
- Method as claimed in claim 1, wherein the synthetic linear peptide corresponds to a sequence of the non-helical C-terminal region of collagen type I.
- Method as claimed in one of the claims 1 and 2, wherein the synthetic linear peptide is composed of 5 to 25 amino acids and preferably of 8 to 20 amino acids.
- Method as claimed in one of the claims 1 to 3, wherein the synthetic peptide corresponds to the sequence shown in SEQ ID NO 1, 2 or 3.
- Standard material for constructing a standard or calibration curve in a competitive immunoassay for the detection of collagen or collagen fragments of type I, wherein it contains an antigen which contains a synthetic linear peptide that corresponds to a sequence of the non-helical C- or N-terminal region of collagen type I.
- Process for the production of the standard material as claimed in claim 5, wherein a linear peptide that corresponds to a sequence of the non-helical C- or N-terminal region of collagen type I is synthesized and coupled optionally via a spacer to a suitable carrier molecule via its N- or C-terminal amino acid.
- Antigen for the production of antibodies against collagen type I or collagen fragments, wherein it contains a synthetic linear peptide that corresponds to a sequence of the non-helical C- or N-terminal region of collagen type I.
- Antigen as claimed in claim 7, wherein the synthetic linear peptide is coupled to a suitable carrier protein.
- Process for the production of the antigen as claimed in claim 7, wherein a linear peptide that corresponds to a sequence of the non-helical C- or N-terminal region of collagen type I is synthesized and coupled by means of its N- or C-terminal amino acid to a carrier protein if desired via a spacer.
- Process for the production of antibodies against collagen type I or collagen fragments, wherein an antigen as claimed in claim 7 or 8 is used for the immunization.
- Antibodies against collagen type I or collagen fragments thereof obtainable by immunization with an antigen as claimed in claim 6 or 7 and isolation of the desired antibody from the serum of the immunized animals or by immortalizing the spleen cells of the immunized animals, cloning those immortalized spleen cells which produce the desired antibody and isolating the antibody from the cloned cells or from their culture supernatant.
- Use of an antigen as claimed in one of the claims 7 or 8 in a method for the detection of type I collagen or collagen fragments in a sample of a body liquid as a measure for the extent of osteolysis.
- Use of an antibody as claimed in claim 11 in a method for the detection of type I collagen or collagen fragments in a sample of a body liquid as a measure for the extent of osteolysis.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4225038 | 1992-07-29 | ||
| DE4225038A DE4225038C2 (en) | 1992-07-29 | 1992-07-29 | Production and use of antibodies against collagen |
| PCT/EP1993/002010 WO1994003813A1 (en) | 1992-07-29 | 1993-07-28 | Immunoassay for detecting collagen or collagen fragments |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| EP0659276A1 EP0659276A1 (en) | 1995-06-28 |
| EP0659276B1 true EP0659276B1 (en) | 1998-01-14 |
| EP0659276B2 EP0659276B2 (en) | 2008-07-30 |
Family
ID=6464371
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP93917678A Expired - Lifetime EP0659276B2 (en) | 1992-07-29 | 1993-07-28 | Immunoassay for detecting collagen or collagen fragments |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0659276B2 (en) |
| JP (1) | JP3360826B2 (en) |
| AT (1) | ATE162311T1 (en) |
| CA (1) | CA2139592A1 (en) |
| DE (2) | DE4225038C2 (en) |
| DK (1) | DK0659276T3 (en) |
| ES (1) | ES2114064T5 (en) |
| FI (1) | FI950378A0 (en) |
| NO (2) | NO950325L (en) |
| WO (1) | WO1994003813A1 (en) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0711415B1 (en) * | 1993-07-28 | 1998-07-22 | Boehringer Mannheim Gmbh | Immunoassay for detecting collagen type i or collagen fragments thereof |
| DK104093D0 (en) * | 1993-09-17 | 1993-09-17 | Osteometer A S | PROCEDURE FOR DETERMINING COLLAGEN FRAGMENTS IN BODY LIQUIDS, TEST KITS AND MEANS FOR EXERCISING THE PROCEDURE AND USING THE PROCEDURE FOR DIAGNOSTICING THE DISEASES OF TABLET METAL |
| GB9506050D0 (en) * | 1995-03-24 | 1995-05-10 | Osteometer A S | Assaying collagen fragments in body fluids |
| AU3746295A (en) | 1994-10-17 | 1996-05-06 | Osteometer Biotech As | Estimation of the fragmentation pattern of collagen in body fluids and the diagnosis of disorders associated with the metabolism of collagen |
| DE59505585D1 (en) * | 1994-12-23 | 1999-05-12 | Roche Diagnostics Gmbh | Antigens and antibodies for the detection of collagen I |
| US5750647A (en) * | 1995-05-19 | 1998-05-12 | Washington Research Foundation | Synthetic peptide analogs of NTx |
| US6107047A (en) * | 1996-03-21 | 2000-08-22 | Osteometer Biotech A/S | Assaying protein fragments in body fluids |
| GB9617616D0 (en) | 1996-08-22 | 1996-10-02 | Osteometer Biotech As | Assaying protein fragments in body fluids |
| ES2160985T3 (en) | 1996-12-09 | 2001-11-16 | Osteometer Biotech As | SANDWICH TYPE TESTS FOR COLLAGEN FRAGMENTS. |
| US6117646A (en) * | 1997-09-22 | 2000-09-12 | Osteometer Biotech A/S | Assaying protein fragments in body fluids |
| US6602980B1 (en) | 1998-06-19 | 2003-08-05 | Washington Research Foundation | Collagen type III synthetic peptides for collagen resorption assays |
| US6916903B2 (en) | 1998-06-19 | 2005-07-12 | Washington Research Foundation | Collagen type III synthetic peptides for collagen resorption assays |
| US6348320B1 (en) | 1998-06-19 | 2002-02-19 | Washington Research Foundation | Cartilage resorption assays measuring type II collagen fragments |
| DK1088228T3 (en) | 1998-06-19 | 2006-01-02 | Washington Res Found | Cartilage resorption assays |
| WO2009035959A2 (en) * | 2007-09-10 | 2009-03-19 | Ge Healthcare Limited | Radiofluorination methods |
| GB201514658D0 (en) * | 2015-08-18 | 2015-09-30 | Nordic Bioscience As | Immunoassay for collagen type VIII sequences |
| CN113866406B (en) * | 2021-10-18 | 2022-09-27 | 深圳上泰生物工程有限公司 | Kit for specifically detecting sugar-deficient transferrin |
| CN118146301A (en) * | 2024-01-04 | 2024-06-07 | 广州白云山花城药业有限公司 | Active peptide for promoting bone cell differentiation and application thereof |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3714634A1 (en) * | 1987-05-02 | 1988-11-17 | Hoechst Ag | METHOD FOR SELECTIVE IMMUNOLOGICAL DETERMINATION OF INTACT PROCOLLAGEN PEPTIDE (TYPE III) AND PROCOLLAGEN (TYPE III) IN BODY LIQUIDS AND MEANS TO IMPLEMENT IT |
| ES2065350T3 (en) * | 1988-04-27 | 1995-02-16 | Hoechst Ag | MONOCLONAL ANTIBODY FOR THE SELECTIVE IMMUNOLOGICAL DETERMINATION OF INTACT AND PROCOLAGEN PEPTIDE (TYPE III) (TYPE III) IN BODY LIQUIDS. |
| WO1990008195A1 (en) * | 1989-01-13 | 1990-07-26 | President And Fellows Of Harvard College | Monoclonal antibody to human type ix collagen |
| US5081031A (en) * | 1989-12-14 | 1992-01-14 | Regents Of The University Of Minnesota | Synthetic polypeptide with type iv collagen activity |
| GB9014220D0 (en) * | 1990-06-26 | 1990-08-15 | Farmos Yhtymy Oy | Method for the immunlolgical determinition of the carboxyterminal propeptide of type i procollagen |
| GB9105893D0 (en) * | 1991-03-20 | 1991-05-08 | Orion Yhtymae Oy | Bone resorption assay based on a peptide liberated during collagen degradation |
| ATE161543T1 (en) * | 1992-01-31 | 1998-01-15 | David Jeston Baylink | AMINOTERMINAL PROCOLLAGEN 1(I) PEPTIDES |
-
1992
- 1992-07-29 DE DE4225038A patent/DE4225038C2/en not_active Expired - Fee Related
-
1993
- 1993-07-28 JP JP50497794A patent/JP3360826B2/en not_active Expired - Fee Related
- 1993-07-28 EP EP93917678A patent/EP0659276B2/en not_active Expired - Lifetime
- 1993-07-28 CA CA002139592A patent/CA2139592A1/en not_active Abandoned
- 1993-07-28 AT AT93917678T patent/ATE162311T1/en not_active IP Right Cessation
- 1993-07-28 WO PCT/EP1993/002010 patent/WO1994003813A1/en active IP Right Grant
- 1993-07-28 ES ES93917678T patent/ES2114064T5/en not_active Expired - Lifetime
- 1993-07-28 DE DE59308000T patent/DE59308000D1/en not_active Expired - Lifetime
- 1993-07-28 DK DK93917678T patent/DK0659276T3/en active
-
1995
- 1995-01-27 FI FI950378A patent/FI950378A0/en unknown
- 1995-01-27 NO NO950325A patent/NO950325L/en not_active Application Discontinuation
-
1996
- 1996-01-26 NO NO960349A patent/NO960349L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| DK0659276T3 (en) | 1998-09-14 |
| FI950378A (en) | 1995-01-27 |
| DE4225038A1 (en) | 1994-02-03 |
| ES2114064T3 (en) | 1998-05-16 |
| NO960349D0 (en) | 1996-01-26 |
| EP0659276A1 (en) | 1995-06-28 |
| EP0659276B2 (en) | 2008-07-30 |
| ES2114064T5 (en) | 2008-11-16 |
| NO960349L (en) | 1996-01-26 |
| FI950378A0 (en) | 1995-01-27 |
| WO1994003813A1 (en) | 1994-02-17 |
| JPH07509778A (en) | 1995-10-26 |
| JP3360826B2 (en) | 2003-01-07 |
| DE4225038C2 (en) | 1995-11-30 |
| DE59308000D1 (en) | 1998-02-19 |
| NO950325D0 (en) | 1995-01-27 |
| NO950325L (en) | 1995-01-27 |
| CA2139592A1 (en) | 1994-02-17 |
| ATE162311T1 (en) | 1998-01-15 |
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